Sex Specific Differences in Response to Calorie Restriction in Skeletal Muscle of Young Rats

Calorie restriction (CR), defined as a reduction of the total calorie intake of 30% to 60% without malnutrition, is the only nutritional strategy that has been shown to extend lifespan, prevent or delay the onset of age-associated diseases, and delay the functional decline in a wide range of species. However, little is known about the effects of CR when started early in life. We sought to analyze the effects of CR in the skeletal muscle of young Wistar rats. For this, 3-month-old male and female rats were subjected to 40% CR or fed ad libitum for 3 months. Gastrocnemius muscles were used to extract RNA and total protein. Western blot and RT-qPCR were performed to evaluate the expression of key markers/pathways modulated by CR and affected by aging. CR decreased body and skeletal muscle weight in both sexes. No differences were found in most senescence, antioxidant, and nutrient sensing pathways analyzed. However, we found a sexual dimorphism in markers of oxidative stress, inflammation, apoptosis, and mitochondrial function in response to CR. Our data show that young female rats treated with CR exhibit similar expression patterns of key genes/pathways associated with healthy aging when compared to old animals treated with CR, while in male rats these effects are reduced. Additional studies are needed to understand how early or later life CR exerts positive effects on healthspan and lifespan.

Introduction
Calorie restriction (CR), defined as a reduction of the total calorie intake by 30% to 60% without malnutrition, has been shown to extend lifespan across a wide range of species [1,2]. Furthermore, CR has been proposed as a robust strategy to delay the functional decline associated with aging thereby preventing the onset of most age-related diseases [1,3]. Several conserved signaling pathways are modulated by CR, including the mechanistic target of rapamycin (mTOR) pathway, the insulin-like growth factor (IGF)-insulin pathway, the AMP-activated protein kinase (AMPK) pathway, the nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2) pathway, or via the modulation of Sirtuins [3,4].
CR is thought to exert a protective role on muscle aging, as it preserves muscle mass and function [5]. Although some reports showed no differences in mitochondrial biogenesis, mitochondrial function was found to be preserved in skeletal muscle of animals under CR [6,7]. Furthermore, CR has been shown to improve proteostasis in skeletal muscle, as it modulates protein degradation to prevent the accumulation of oxidative-damaged proteins [8,9]. The attenuation of oxidative stress damage has also been reported in rat cardiac muscle, as well as activation of autophagy with CR [10]. In addition to a reduction in oxidative stress, inflammation levels have also been found reduced in the skeletal muscles of animals under CR [9,11].
However, little is known about the effects of CR when started early in life. Some studies have reported no differences in mitochondrial function or antioxidant enzymatic activities [12], in oxidative metabolism [13], or in mTOR signaling and protein degradation [14] in young rats subjected to CR. Therefore, the aim of this study was to analyze the effects of CR in the skeletal muscle of young Wistar rats. For this, male and female rats aged 3 months were subjected to 40% CR or fed ad libitum for 3 months. Following treatment, gastrocnemius muscles were used to determine the levels of markers of mitochondrial function, antioxidant, autophagy, nutrient-sensing, inflammation, and senescence pathways.

Animals and Diets
All experiments were performed according to general guidelines for animal care approved by the institutional ethics committee and EU regulations (2010/63/UE). Female and male Wistar rats aged 3 months were purchased from Charles River Laboratories (Barcelona, Spain) and housed at 22 • C and 65 ± 3% humidity with a 12-h light/dark cycle. Animals were divided into four experimental groups with different diets for 12 weeks. One group of females (n = 8) and one group of males (n = 3) were fed ad libitum with a pelleted standard diet (A04, Panlab, Barcelona). Another group of females (n = 8) and of males (n = 4) were subjected to 40% CR. Food for CR treated-rats consisted of the same standard diet but was adjusted weekly and dependent on the average food consumed by the same sex ad libitum group, which was used as a reference for baseline food consumption. This was done to correct for growth requirements and avoid delays in development.
At 24 weeks of age, rats were sacrificed by decapitation at the beginning of the light cycle and tissues were collected, weighed, immediately frozen in liquid nitrogen and stored at −80 • C until further analyses. Serum glucose and triglycerides were measured using an Accutrend ® GCT-meter (Roche Diagnostics, Basel, Switzerland).

Western Blot Analysis
For each blot, 40 µg of total protein was separated on SDS-PAGE gels and electrotransferred to 0.22 µm nitrocellulose membranes using the Transblot Turbo transfer system (Bio-Rad). Ponceau S staining was used as a loading control. The intensity of each lane was measured, and the quantification of each protein was corrected to the total amount of protein loaded for each sample. Membranes were then blocked with 5% non-fat powdered milk in Tris Buffer Saline, pH 7.6 (TBS) with 0.05% Tween-20 for 1 h. Antibodies against proteins analyzed are listed in Supplemental Table S1. After overnight incubation with primary antibodies, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase for 1 h and protein bands were detected with Immun-Star™ WesternC™ Chemiluminiscent Kit (Bio-Rad) Western. The chemiluminiscence signal was acquired with a Chemidoc XRS densitometer (Bio-Rad) and results were analyzed with Quantity One Software (Bio-Rad).

Determination of ATPase Enzymatic Activity
Gastrocnemius muscles were homogenized in sucrose buffer (40 mM sucrose, 220 mM mannitol, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) with a Teflon/glass homogenizer. Samples were then centrifuged at 600× g for 10 min at 4 • C and supernatants were recovered. Protein content was analyzed with a Bradford assay and the enzymatic activity of Complex V (ATPase, EC 3.6.1.3) was performed immediately after. Briefly, samples were incubated in a 96-well spectrophotometric plate with the assay buffer (0.33 M sucrose, 6.3 mM MgSO 4 , 63.66 mM HEPES, 0.442 mM NADH, pH 8.0) with 2.5 mM phosphor(enol)pyruvic acid, 0.5 µg/mL Pyruvate Kinase, 0.25 µg/mL L-Lactic dehydrogenase and 0.1 µg/mL antimycin. To start the reaction, 5 mM ATP was added into each well. Oxidation of NADH at 340 nm was followed for 15 min at 37 • C. The generated slope is inversely proportional to the activity of Complex V. The enzymatic activity was expressed as international units (I.U.) per mg of protein, with an extinction coefficient used of 6.22 mM −1 cm −1 .

RT-qPCR
Gastrocnemius muscles (25 mg) were pulverized in liquid nitrogen as previously described and 1 mL of Tri Reagent ® (Sigma-Aldrich, St. Louis, Missouri, United States) was added to each sample. RNA was isolated following manufacturer's instructions and quantified using a BioSpec-nano spectrophotometer (Shimadzu Biotech, Kyoto, Japan).
Total RNA (1 µg) was reverse transcribed to cDNA at 37 • C for 50 min with 200 U M-Mlv reverse transcriptase in a 20 µL volume of reaction mixture containing 1X First-Strand Buffer, 2.5 µM random hexamers, 500 µM each dNTP, 20 U RNAse inhibitor, and 10 mM DTT. Samples were diluted 1/10 and stored at −20 • C until use.
PCR reactions were performed using SYBR Green chemistry on a LightCycler ® 480 System (Roche, Barcelona, Spain). Target genes, primers, and annealing temperatures are specified in Supplemental Table S2. The reaction mixture contained 7.5 µL SYBR TB Green ® Premix Ex Taq™ (RR420A, Takara, Shiga, Japan) with 0.5 µM of forward and reverse primers) and 2.5 µL of cDNA. The amplification program included a denaturation step at 95 • C, 5 min, followed by 45 cycles with a denaturation step (10 s, 95 • C), an annealing step (10 s, temperature depending on primers, see Supplemental Table S2), and an elongation step (12 s, 72 • C). A negative control without cDNA was run in each assay. Ct values obtained were analyzed, considering the efficiency of each reaction, using the GenEx Standard Software (Multi-DAnalises, Göteborg, Sweden). Two housekeeping genes, Rpl32 and Tbp, were used according to the Normfinder algorithm.

String Analysis
Protein-protein interactions (PPIs) were analyzed using the STRING database (v.11.5; www.string-db.org, accessed on 24 August 2022) by querying genes that were differentially expressed genes and proteins obtained in the results. The network edges represent either the evidence of the PPI or the confidence. All active interaction sources were selected, and the minimum required interaction score was set at medium confidence (0.400).

Statistical Analysis
Results are presented as mean ± Standard error of the mean (SEM) unless otherwise specified. Differences between CR and control groups were assessed by Student's t-test, and interaction effects between sexes were generated by two-way analysis of variance (ANOVA) and post hoc Tukey's test. A p value < 0.05 was considered statistically significant. To test potential associations between the measured parameters, a principal component analysis (PCA) was computed. Statistical analyses were performed using R Studio version 3.5.2 of the R programming language (R Project for Statistical Computing; R Foundation, Vienna, Austria).

CR Reduced Body and Tissue Weights
Body mass, tissue weights, and serum levels of glucose and triglycerides of control and CR male and female rats are shown in Table 1. Body weight was significantly decreased in animals subjected to CR compared to control animals. Both sexes exhibited similar weight loss, −27.1% in females, and −31.3% in males. Treatment with CR also reduced the weight of adipose tissue (i.e., inguinal, lumbar, mesenteric, and ovarian/epididymal adipose tissues) and the liver (−62.2% and −31.3% in females, and −67.0% and −33.2% in males, respectively). Skeletal muscle weight was lower in female rats compared to their male counterparts and CR reduced muscle weight similarly in both sexes. However, the muscle somatic index (MSI), obtained by adjusting muscle skeletal weight to body mass, was increased in both male and female CR rats, suggesting that skeletal muscle specifically is preserved with CR in young animals. In fact, total protein content of skeletal muscle tissue remained unchanged with diet ( Figure 1A-C). Finally, serum glucose was unaffected by diet, while triglycerides were reduced by 50% but this effect was seen only in male rats subjected to CR. Finally, principal component analysis (PCA) showed a clear separation between female and male rats treated with CR ( Figure 1D).

CR Effects on Mitochondrial Function and Dynamics
To evaluate the effects of early onset CR on mitochondrial bioenergetics in skeletal muscle, protein levels of the oxidative phosphorylation (OXPHOS) complexes were analyzed by Western Blot (Table 2, Figure 2), as proteins involved in the main bioenergetic function of these organelles. Complex I and V showed no differences with CR regardless of the sex, while complexes II, III, and V were increased in CR but only female rats. On the other hand, complex III was decreased with CR, but this effect was seen only in male rats. Importantly, Complex IV/Complex V ratio remained unvaried with CR in both sexes. Mitochondrial dynamics were also evaluated via the analysis of mitochondrial fission- performed on all data including weights, mRNA and protein expression (excluding mitochondrial complexes). The first component explains~18% of the variation, and the second component~15%. Two-way ANOVA was performed to assess for significance. Abbreviations: S, sex differences; D, diet differences; N, non-significant; PC1, principal component 1; PC2: principal component 2. * Significant difference between calorie-restricted and control group. Table 1. Body and tissue weights, glucose and triglycerides levels in control and restricted rats. Two-way ANOVA was performed to assess for significance. Abbreviations: S, sex differences; D, diet differences; SxD, interactive effect between sex and diet; NS, non-significant. WAT weight: represents sum of inguinal, lumbar, mesenteric, epididymal/ovarian adipose tissues weights. * Statistical differences between calorie restricted and control groups.

CR Effects on Mitochondrial Function and Dynamics
To evaluate the effects of early onset CR on mitochondrial bioenergetics in skeletal muscle, protein levels of the oxidative phosphorylation (OXPHOS) complexes were analyzed by Western Blot (Table 2, Figure 2), as proteins involved in the main bioenergetic function of these organelles. Complex I and V showed no differences with CR regardless of the sex, while complexes II, III, and V were increased in CR but only female rats. On the other hand, complex III was decreased with CR, but this effect was seen only in male rats. Importantly, Complex IV/Complex V ratio remained unvaried with CR in both sexes. Mitochondrial dynamics were also evaluated via the analysis of mitochondrial fission-related proteins GTPase dynamin-related protein 1 (DRP1) and mitochondrial fission 1 protein (FIS1) levels. Interestingly, there were not any differences in levels of these proteins in female rats subjected to CR, but male rates undergoing CR exhibited significant decreases in both DRP1 and FIS1 expression levels ( Figure 3). Table 2. Protein levels of OXPHOS complexes and mitochondrial fission proteins analyzed by Western Blot. Two-way ANOVA was performed to assess for significance. Abbreviations: S, sex differences; D, diet differences; N, non-significant. * Statistical differences between calorie restricted and control groups.    Complex V or ATPase enzymatic activity was measured to obtain an indirect measure of skeletal muscle function. ATPase activity remained unchanged with CR in both sexes (p-value = 0.429) ( Figure S1). We observed a significant sex difference in ATPase activity (p-value < 0.01), with females showing twice the activity as males. Table 3 shows the mRNA levels and Table 4 the protein levels of several key antioxidant proteins that are involved in aging and modulated by CR in older age. Interestingly, in younger rats, we were not able to detect differences in most of these genes/proteins but there was an increase in the level of sirtuin 3 (SIRT3) protein in female CR rats (Figure 4). Interestingly, no changes were observed in acetylated levels of   Complex V or ATPase enzymatic activity was measured to obtain an indirect measure of skeletal muscle function. ATPase activity remained unchanged with CR in both sexes (p-value = 0.429) ( Figure S1). We observed a significant sex difference in ATPase activity (p-value < 0.01), with females showing twice the activity as males. Table 3 shows the mRNA levels and Table 4 the protein levels of several key antioxidant proteins that are involved in aging and modulated by CR in older age. Interestingly, in younger rats, we were not able to detect differences in most of these genes/proteins but there was an increase in the level of sirtuin 3 (SIRT3) protein in female CR rats ( Figure 4). Interestingly, no changes were observed in acetylated levels of superoxide dismutase 2 (SOD2), a known target of SIRT3. On the other hand, NRF2 expression levels, a key transcription factor involved in antioxidant pathways, were different between males and females but not influenced by CR (Figure 4). Table 3. mRNA expression levels of antioxidant genes. Two-way ANOVA was performed to assess for significance. NS: non-significant. Sod1: superoxide dismutase 1; Sod2: superoxide dismutase 2; sirt3: sirtuin 3; Nfe2l2: nuclear factor erythroid 2-related factor 2; Foxo3: forkhead box O3.

Autophagy and Apoptosis
mRNA levels of Map1lc3a and Sqstm1 (Table 5), and protein levels of LC3 and Caspase 3 (Table 6) were measured as key makers of autophagy and apoptosis. No differences among groups were observed in Map1lc3a and Sqstm1 gene expression, and no changes were found in LC3A/B protein levels or the LC3-II/LC3-I ratio. However, we did detect interactive effects between sex and diet for caspase 3 and noted significant decreases in caspase 3 protein expression levels, but only in male rats subjected to CR ( Figure 5).

Autophagy and Apoptosis
mRNA levels of Map1lc3a and Sqstm1 (Table 5), and protein levels of LC3 and Caspase 3 ( Table 6) were measured as key makers of autophagy and apoptosis. No differences among groups were observed in Map1lc3a and Sqstm1 gene expression, and no changes were found in LC3A/B protein levels or the LC3-II/LC3-I ratio. However, we did detect interactive effects between sex and diet for caspase 3 and noted significant decreases in caspase 3 protein expression levels, but only in male rats subjected to CR ( Figure 5).    Table 7 shows the mRNA levels of several key genes related to nutrient-sensing pathways. Interestingly, there were no differences between groups/sex in expression levels of Pik3ca, Akt1, Gsk3b, and mTOR. Only the expression of Hif1a showed an interactive effect between sex and diet and this effect was significantly reduced in male rats subjected to CR ( Figure 6A).  Table 7 shows the mRNA levels of several key genes related to nutrient-sensing pathways. Interestingly, there were no differences between groups/sex in expression levels of Pik3ca, Akt1, Gsk3b, and mTOR. Only the expression of Hif1a showed an interactive effect between sex and diet and this effect was significantly reduced in male rats subjected to CR ( Figure 6A). Table 7. mRNA expression levels of key autophagy markers. Two-way ANOVA was performed to assess for significance. S indicates sex effect; SxD indicates interactive effect between sex and diet. NS: non-significant; * Significant differences between calorie-restricted and control groups. Pik3ca: phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha; Akt1: AKT serine/threonine kinase 1; Gsk3b: glycogen synthase kinase 3 beta; Mtor: mechanistic target of rapamycin kinase; Hif1a: hypoxia inducible factor 1 subunit alpha.

Inflammation and Senescence
Levels of key mediators of inflammation and senescence are shown in Tables 9-11. No changes were observed in mRNA levels of Tnf and TGFb, while Il1b ( Figure 7A) expression increased but only in male rats subjected to CR, while interleukin 1 receptor (IL1R) protein levels remained unvaried by diet. Interleukin 6 (IL-6), a key protein involved in inflammatory response, ( Figure 7B) increased with CR only but this effect was As seen in Table 8, protein levels of insulin receptor beta (IRb) and insulin receptor substrate (IRS) were not influenced by CR or sex. Similarly, protein kinase B (AKT), liver kinase B1 (LKB1), and lactate dehydrogenase (LDH) were unaffected by diet or sex. Interestingly, only levels of estrogen-related receptor alpha (ERRα) were increased in male and female rats treated with CR ( Figure 6B,C). Both AMPK and glycogen synthase kinase (GSK) showed a tendency (p = 0.07 and p = 0.05, respectively) to decrease in females rats subjected to CR. Finally, CR was associated with a decrease in isocitrate dehydrogenase 2 (IDH2) protein levels but this effect was only seen in males. Table 8. Protein levels of several nutrient-sensing pathways markers analyzed by Western Blot. Two-way ANOVA was performed to assess for significance. D indicates diet differences. NS: nonsignificant. * Statistical differences between calorie restricted and control groups. All data were normalized to ad libitum females.

Inflammation and Senescence
Levels of key mediators of inflammation and senescence are shown in Tables 9-11. No changes were observed in mRNA levels of Tnf and TGFb, while Il1b ( Figure 7A) expression increased but only in male rats subjected to CR, while interleukin 1 receptor (IL1R) protein levels remained unvaried by diet. Interleukin 6 (IL-6), a key protein involved in inflammatory response, ( Figure 7B) increased with CR only but this effect was only noted in females. On the other hand, NF-kB mRNA expression did not change with CR, but interestingly protein levels of nuclear factor kappa b (NF-kB) ( Figure 7C) increased with CR in females. Inhibitor of nuclear factor kappa b (IkB) protein levels ( Figure 7D) decreased with CR but this effect was noted only in males. We did not find any changes in Tp53, Cdkn2a, and Sirt6 mRNA levels among groups, and only a tendency (p = 0.07) of increased expression of Cdkn1a in male rats subjected to CR. P53 protein levels were also unaffected by diet or sex. Table 9. mRNA expression levels of inflammatory markers. Two-way ANOVA was performed to assess for significance. NS: non-significant. * Statistical differences between calorie restricted and control groups. Rela: RELA proto-oncogen, nuclear factor kappa B subunit; Il1b: interleukin 1 beta; Tgfb1: transforming growth factor beta 1; Tnf: tumor necrosis factor.

Analysis of Protein-Protein Interaction
Finally, we analyzed possible protein-protein interactions using the STRING database. Figure 8A shows the main interactions and the interaction type found by querying the genes and proteins that were significantly affected by CR. The PPI enrichment p-value for this network was 5.05 × 10 −11 , with 24 edges. Specific networks for females and males were also constructed. In Figure 8B,C, the network with the

Analysis of Protein-Protein Interaction
Finally, we analyzed possible protein-protein interactions using the STRING database. Figure 8A shows the main interactions and the interaction type found by querying the genes and proteins that were significantly affected by CR. The PPI enrichment p-value for this network was 5.05 × 10 −11 , with 24 edges. Specific networks for females and males were also constructed. In Figure 8B,C, the network with the differentially expressed genes and proteins for females and males are shown, respectively. The thickness of the edges represents the strength of data supporting each PPI. The PPI enrichment values for these networks were 0.0103 for females and 0.000826 for males.

Discussion
In this study, we analyzed the influence of 40% CR on markers of skeletal muscle function in 3-months old male and female Wistar rats. As expected, treatment with CR resulted in a decrease in body, liver, adipose tissue, and skeletal muscle weights. We also measured the expression of some key age-related markers related to the hallmarks of aging and involved in mitochondrial biology, antioxidant activity, autophagy and apoptosis, nutrient-sensing pathways, inflammation, and senescence. Interestingly, we found significant differences among dietary treatment and sex in only some mitochondrial-related proteins and in a few key markers of antioxidant, apoptosis, inflammatory and nutrient-sensing pathways.
In the current study, CR reduced total body weight, liver weight, fat mass, as well as muscle mass, which is in accordance with what has been previously reported [12,13,15,16]. However, MSI, an index of sarcopenia [17,18], and the total protein content of skeletal muscle, were increased with CR in both males and females. These results are agreement with previous observations in the tibialis cranialis and the soleus muscles from rats under to energy restriction [17]. Furthermore, Colom et al. [12] also reported conserved MSI and protein content in gastrocnemius muscles in male CR rats, and even an increase in protein content in female CR rats. Conversely, Miller et al. [19] showed that the synthesis of mitochondrial protein rate was similar in skeletal muscle of rats, regardless of age and CR treatment. These results, in addition to the results in the current study, indicate that skeletal muscle function might be preserved by CR in young male and female rats.

Discussion
In this study, we analyzed the influence of 40% CR on markers of skeletal muscle function in 3-months old male and female Wistar rats. As expected, treatment with CR resulted in a decrease in body, liver, adipose tissue, and skeletal muscle weights. We also measured the expression of some key age-related markers related to the hallmarks of aging and involved in mitochondrial biology, antioxidant activity, autophagy and apoptosis, nutrient-sensing pathways, inflammation, and senescence. Interestingly, we found significant differences among dietary treatment and sex in only some mitochondrial-related proteins and in a few key markers of antioxidant, apoptosis, inflammatory and nutrientsensing pathways.
In the current study, CR reduced total body weight, liver weight, fat mass, as well as muscle mass, which is in accordance with what has been previously reported [12,13,15,16]. However, MSI, an index of sarcopenia [17,18], and the total protein content of skeletal muscle, were increased with CR in both males and females. These results are agreement with previous observations in the tibialis cranialis and the soleus muscles from rats under to energy restriction [17]. Furthermore, Colom et al. [12] also reported conserved MSI and protein content in gastrocnemius muscles in male CR rats, and even an increase in protein content in female CR rats. Conversely, Miller et al. [19] showed that the synthesis of mitochondrial protein rate was similar in skeletal muscle of rats, regardless of age and CR treatment. These results, in addition to the results in the current study, indicate that skeletal muscle function might be preserved by CR in young male and female rats.
Mitochondrial dysfunction, including alterations in the OXPHOS complexes and in mitochondrial dynamics, is as a key hallmark of aging [2,20,21]. In skeletal muscle, CR has shown to preserve mitochondrial function by increasing OXPHOS activity or mitochondrial respiration [7,12]. In the present study, we observed a slight increase in the protein level of several subunits of the oxidative phosphorylation complexes in females rats subjected to CR, although the complex IV/complex V ratio remained unvaried, thus indicating a preserved mitochondrial oxidative capacity. On the contrary, there was a decrease in the levels of complex III with CR, but this effect was only seen in male rats. Unfortunately, we could not determine the enzymatic activities of the OXPHOS complexes, which could have revealed more valuable information about mitochondrial bioenergetics. Previous studies conducted in young male rats showed no changes in OXPHOS levels or function with CR [6,22], although Wang et al. [23] suggested that the effect of CR on mitochondrial function might be fiber-type dependent. On the other hand, Gutiérrez-Casado et al. [24] reported a decrease in complex III in the skeletal muscle of mice subjected to 18 months of CR. In the current study, we observed a decrease in both DRP1 and FIS1 fission-related proteins in male rats treated with CR. Mitochondrial fission is thought to be induced in aged skeletal muscle, and long-term CR has been shown to reduce the levels of FIS1 and DRP1 [18,25]. These results suggest that fission is attenuated by CR, presumably to stimulate mitochondrial fusion or to avoid mitochondrial degradation [26]. Interestingly, in the current study, we did not observe any changes in key autophagy markers, which seem to be induced in older animals subjected to CR [24].
CR has also the potential to modulate satellite cells, the stem cells of skeletal muscle, which are involved in muscle homeostasis and regeneration [27,28]. Some reports suggest that CR increases the number of satellite cells and enhances their regenerative ability [27,29,30]. Cerletti et al. [29] showed that satellite cells from young mice under CR were metabolically reprogrammed and presented increased number of mitochondria and oxidative phosphorylation. However, Abreu et al. [30] reported that the increased proliferation of satellite cells of CR mice is not consequence of a change in metabolism.
Oxidative stress is closely related to mitochondrial dysfunction and also plays a role in aging [26]. CR has been reported to reduce oxidative damage in several tissues and animal models [7,10,25,31]. In our study, SIRT3 was increased in females under CR, although other antioxidant proteins were unvaried. This limited effect could be explained because animals were too young to 'consider' oxidative stress a determinant factor. In fact, previous studies have shown that the enzymatic activity of some antioxidant proteins are preserved in young skeletal muscle and these antioxidant defenses were higher in females than in males [12]. This result is consistent with our findings showing increased levels of NRF2 in females.
Interestingly, we found that protein levels of caspase 3 was decreased in male rats under CR and was unvaried in females. Caspase 3 has been reported to be involved in muscle differentiation and the promotion of myogenesis [32,33]. In fact, this protein regulates multiple pathways involved in skeletal muscle growth. Caspase 3 is responsible for the cleavage of Pax7, a transcription factor regulating self-renewal of satellite cells [34], the stem cells of skeletal muscle. Thus, this decrease in caspase 3 would indicate a preservation of stem cell niche in males. However, other factors should be considered to further analyze the effects of CR on the satellite cell population in younger rats, including the transcription factor NF-Y [28]. On the other hand, caspase 3 also regulates p21 expression, which results in cell cycle arrest and the initiation of the cell's differentiation program [34]. In this regard, we only observed a slight trend of increased Cdkn1a mRNA expression levels in males. The function of skeletal muscle was preserved in both males and females, as discussed previously and based on the MSI and total skeletal muscle protein content.
Very few changes were observed in genes and proteins related to nutrient-sensing pathways, which is in agreement with other animal studies, suggesting that the benefits of CR are limited in younger animals whose muscle function is adequate [13,14]. In the current study, Hif1a mRNA expression was significantly reduced but only in males under CR. HIF1, an hypoxia-inducible factor, mediate transcriptional responses to hypoxia and has been shown to increase with aging, and CR is able to attenuate this effect [35]. On the other hand, in the current study, ERRα was increased with CR, perhaps due to its involvement in the regulation of mitochondrial function and cellular metabolism [36].
In our study, contradictory results were also found when analyzing the effect of sex and diet on key inflammatory markers. Male rats under CR showed increased expression of IL1b and decreased levels of IκB, a negative regulator of the transcription factor NF-κB [35]. These results suggest that there is an increase in inflammation when CR is started at a younger age. On the contrary to the results in males, there was a decrease in IL6 and NF-κB protein levels in younger CR treated female rats. Previous reports have shown that CR decreases key inflammatory mediators in several tissues, including skeletal muscle [11,35,37,38]. It should be noted that most of our results are indirect measures of inflammation. For instance, in the current study, only an increase in the IL1b mRNA expression was observed, which does not necessarily translate into higher protein levels of this cytokine. Furthermore, no downstream effects of IκB and NF-κB were observed. The decrease in IL6 levels in female CR treated rats are in agreement with results from previous studies [11,33]. Finally, we were not able to detect any changes in markers of senescence among the groups. As mentioned before, this may indicate an attenuated effect or a ceiling effect of CR in young animals, as age-related senescence is not yet a determinant factor in these animals.
Skeletal muscle has been shown to directly modulate lifespan in several species. For instance, the overexpression of Atg8, a gene involved in autophagy, in fly muscles is sufficient to extend their lifespan [39]. CR can have an impact on skeletal muscle health, improving processes such as autophagy, proteostasis, antioxidant defenses, or mitochondrial function. Through the release of myokines and several metabolites, skeletal muscle can affect other tissues including the brain, the adipose tissue, or the liver [40]. Furthermore, avoiding the loss of skeletal muscle delays age-related diseases such as diabetes, obesity, or some types of cancer, and reduces chronic inflammation and insulin resistance [41]. Alternative to CR, maternal obesity and post-natal over nutrition have been reported to impair skeletal muscle function via glucose and lipid metabolism mechanisms, highlighting muscle metabolic dysfunction as a risk factor to develop insulin resistance and diabetes [42]. Thus, maintaining healthy, functional skeletal muscles can contribute to maintain organism homeostasis and delay aging, improving lifespan.
This study has limitations. First, the sample size was small and therefore there may be limited statistical power to detect differences in gene or protein expression. Second, the treatment length was only of 3 months and therefore the identification of biological pathways affected by CR starting early in life, and how these pathways are related to lifespan, cannot be determined. Finally, our findings are exploratory and therefore it is not possible to make causal conclusions about the effects of early onset CR on muscle health. We did not perform any functional measures of skeletal muscle function, such as grip strength. Some studies in CR-animals suggest that this intervention can enhance grip strength in both young (8 months) and old rats (37 months) [43,44]. However, a recent report shows that CR may increase relative grip strength during the first months because of the reduced body weight, although there were no differences in older animals [45]. On the other hand, using the same rat model, our group has already described that energy expenditure and energy efficiency decreased in the first weeks of CR, but became similar to the control group from the fifth week of intervention [16]. It would have also been of interest to check the effect of CR on muscle fiber type in early life, although the collection method was not adequate to perform any histological analyses. In this regard, other authors have shown that CR in older rats prevents fiber loss [46] and does not change fiber type composition, although CR preserved the mitochondrial morphology only in oxidative fibers [18]. Despite these limitations, our study provides convincing evidence that CR started early in life influences some key genes/proteins involved in aging and importantly, these effects are largely sex-dependent.

Conclusions
In summary, our results suggest a sexual dimorphism in markers of oxidative stress, inflammation, apoptosis, and mitochondrial function in response to CR in young rats. Furthermore, our data show that young female CR treated rats exhibit similar expression patterns of key hallmarks of muscle aging to those observed in older animals under CR, which are related to improved helthspan and lifespan (prolongevity effects). Interestingly, these effects are reduced in younger male CR treated rats. A 'ceiling effect' for CR may be present in younger animals, although no adverse effects were reported in skeletal muscle. Future studies are needed to understand whether and how early or late life CR may exert positive effects on healthspan and lifespan in multiple tissues.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.


s2fieldsofstudy: Biology Medicine

text:
SU16f inhibits fibrotic scar formation and facilitates axon regeneration and locomotor function recovery after spinal cord injury by blocking the PDGFRβ pathway

Background Excessively deposited fibrotic scar after spinal cord injury (SCI) inhibits axon regeneration. It has been reported that platelet-derived growth factor receptor beta (PDGFRβ), as a marker of fibrotic scar-forming fibroblasts, can only be activated by platelet-derived growth factor (PDGF) B or PDGFD. However, whether the activation of the PDGFRβ pathway can mediate fibrotic scar formation after SCI remains unclear. Methods A spinal cord compression injury mouse model was used. In situ injection of exogenous PDGFB or PDGFD in the spinal cord was used to specifically activate the PDGFRβ pathway in the uninjured spinal cord, while intrathecal injection of SU16f was used to specifically block the PDGFRβ pathway in the uninjured or injured spinal cord. Immunofluorescence staining was performed to explore the distributions and cell sources of PDGFB and PDGFD, and to evaluate astrocytic scar, fibrotic scar, inflammatory cells and axon regeneration after SCI. Basso Mouse Scale (BMS) and footprint analysis were performed to evaluate locomotor function recovery after SCI. Results We found that the expression of PDGFD and PDGFB increased successively after SCI, and PDGFB was mainly secreted by astrocytes, while PDGFD was mainly secreted by macrophages/microglia and fibroblasts. In addition, in situ injection of exogenous PDGFB or PDGFD can lead to fibrosis in the uninjured spinal cord, while this profibrotic effect could be specifically blocked by the PDGFRβ inhibitor SU16f. We then treated the mice after SCI with SU16f and found the reduction of fibrotic scar, the interruption of scar boundary and the inhibition of lesion and inflammation, which promoted axon regeneration and locomotor function recovery after SCI. Conclusions Our study demonstrates that activation of PDGFRβ pathway can directly induce fibrotic scar formation, and specific blocking of this pathway would contribute to the treatment of SCI.

astrocytes after SCI, fibrotic scar formed by fibroblasts is less well understood [2,3]. Following SCI, perivascular fibroblasts leave the blood vessel, proliferate, migrate and deposit fibrous extracellular matrix (ECM), including fibronectin, laminin and collagen, finally forming fibrotic scar inside the astrocytic scar, which hinders axon regeneration [4][5][6][7]. Moderate inhibition of fibrotic scar formation contributes to axon regeneration and locomotor function recovery, indicating that the adverse effects of excessively deposited fibrotic scar are greater than the beneficial effects after SCI [6,8]. These results reveal that the role of fibrotic scar in the targeted therapy of SCI is of great significance and should be given more attention. However, the molecular mechanism of fibrotic scar formation after SCI is unclear.
Platelet-derived growth factors (PDGFs) are a cysteineknot-type growth factor family composed of four polypeptide chains A, B, C, and D [9]. These growth factors activate intracellular signalling by binding to plateletderived growth factor receptor (PDGFR) α or PDGFRβ, while PDGFRβ can only be activated by PDGFB and PDGFD and plays an important role in cell proliferation, differentiation and migration [10][11][12]. A large number of studies have shown that the PDGF/PDGFR pathway is a critical functional mediator of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS), while relatively little is known about it in SCI [12]. It has been reported that PDGFRβ, as a marker of perivascular fibroblasts, is expressed in almost all scar-forming fibroblasts [5,6,13], while whether the PDGFRβ pathway is involved in fibrotic scar formation after SCI is still lacking direct evidence. Our previous in vitro study showed that PDGFB can regulate the migration of fibrotic scarforming cells model PDGFRβ + pericytes/fibroblasts, which can be aborted by the PDGFRβ inhibitor SU16f [14]. Nevertheless, the cellular location and function of the PDGF/PDGFRβ pathway after SCI need to be further explored in vivo.
In this study, our results showed that the expression of PDGFD occurred earlier than that of PDGFB after SCI, and PDGFB was mainly secreted by astrocytes, while PDGFD was mainly secreted by macrophages/microglia and fibroblasts. Intrathecal injection of the PDGFRβ inhibitor SU16f blocked the fibrosis induced by exogenous PDGFB or PDGFD in the uninjured spinal cord. In addition, SU16f blockade of the PDGFRβ pathway resulted in the reduction and interruption of fibrotic scar and the resolution of lesion and inflammation, thereby facilitating axon regeneration and locomotor function recovery after SCI. These results indicate that the PDGFRβ pathway is essential for fibrotic scar formation after SCI and is expected to be a therapeutic target for SCI.

Animals and spinal cord compression injury model
All experiments involving animals were approved by the Ethics Committee of Anhui Medical University (Approval No. LLSC20160052). Eight-week-old C57BL/6 mice were acquired from the Animal Experiment Center of Anhui Medical University and were housed in an environment with controlled temperature and humidity and a 12:12 h light:dark cycle. The animals were randomly grouped and kept in standardized cages, where water and food were readily available.
The establishment of the spinal cord compression injury model has been described in detail in our previous study [14]. In brief, after satisfactory anaesthesia with isoflurane (induction 4%, maintenance 2%), the mid-thoracic level (T10) spinal cord was carefully exposed and compressed with calibrated Dumont #5 forceps (11252-20, Fine Science Tools, Germany) for 5 s. The postoperative mice received anti-infection treatment and auxiliary urination nursing twice a day.

In situ injection of PDGFB or PDGFD
The object of in situ injection of PDGFB was the uninjured spinal cord of mice. The T10 spinal cord was exposed according to the established method of the spinal cord injury model, and then the mouse was fixed on the stereotaxic device. The insertion site of the microinjection needle (7634-01 and 7803-05, Hamilton, Switzerland) was 0.3 mm lateral to the midline and 0.8 mm deep to the dorsal surface of the mouse spinal cord [15]. Two microlitres of 100 ng/μl recombinant human PDGFB (HZ-1308, Proteintech, China) dissolved in 10 mM HOAc containing 0.1% bovine serum albumin (BSA) or PDGFD (1159-SB/CF, R&D Systems, United States) dissolved in 4 mM HCl containing 0.1% BSA was injected into the uninjured spinal cord at 0.5 μl/min using a stereotaxic injector (KDS LEGATO 130, RWD, China). The control mice received 2 μl of 10 mM HOAc containing 0.1% BSA or 4 mM HCl containing 0.1% BSA. All mice were sacrificed at 7 days after injection.

Intrathecal injection of SU16f
The needle insertion site was located in the dorsal midpoint of the lumbar 5-6 intervertebral space as previously reported [16]. It was confirmed that the needle was successfully inserted into the intradural space by observing an evident sudden tail flick. Ten microlitres of 3 mM SU16f dissolved in 0.1 M phosphate buffered saline (PBS) containing 3% DMSO (3304, R&D Systems, United States) was injected daily at 1 μl/4 s using a microinjection needle (1701, Hamilton, Switzerland). For the mice without SCI, SU16f was preinjected the day before the injection of PDGFB or PDGFD and then injected daily for 7 consecutive days from the day of PDGFB or PDGFD injection. For the mice with SCI, SU16f was injected from 3 day post-injury (dpi) until sacrifice. The control mice received 10 μl of PBS containing 3% DMSO.

Tissue preparation and immunofluorescent staining
After cardiac perfusion with 0.1 M PBS (Servicebio, China) followed by 4% paraformaldehyde (PFA, Servicebio, China), the 0.5 mm segment of spinal cord tissue containing the injured core was placed in 4% PFA and postfixed for 5 h. The tissue was then placed in a 30% sucrose solution and dehydrated at 4 °C for 24 h until the tissue sank to the bottom. Finally, the tissue was cut into 18 μm-thick serial sagittal or coronal sections using a cryostat (NX50, Thermo Fisher Scientific, United States). The sections encompassing the lesion core or injection site were used.
For BrdU staining, the sections were pretreated with 2 N hydrochloric acid (HCl, GEMIC, China) at 37 °C for 30 min followed by 0.1 M borate buffer (KGR0101, Key-GEN BioTECH, China) at room temperature for 10 min and were subjected to an immunofluorescence staining protocol. The sections were blocked in 10% donkey serum containing 0.3% Triton X-100 (SL050 and T8200, Solarbio, China) at room temperature for 1 h, followed by incubation with primary antibodies at 4 °C overnight. The primary antibodies included goat anti-PDGFRβ

Image acquisition and quantitative analysis
Representative images of the sections were acquired using a Zeiss LSM 900 confocal microscope system and a Zeiss Axio Scope A1 fluorescence microscope. Staining colocalization was determined using ZEN 3.3 software to examine each of the ten one-micron Z-stack slices. Image processing was performed using ImageJ version 2.0 (NIH, United States).
All quantitative analyses were performed in a blind fashion. To quantify GFAP + , CD68 + , CD31 + , PDGFRβ + , PDGFB + and PDGFD + cells, 100 μm square grids were generated over the injured site [17]. Every 6th square was quantified, and only DAPI + cells were counted. One section encompassing the lesion core in each sample was used for counting, with 5 samples per group.
To evaluate the area of fibrotic scar, the immunoreactivities of PDGFRβ, fibronectin and laminin were normalized to the area of the spinal cord segment spanning the injured core in a 4 × image [17]. Similarly, the GFAP − area and CD68 + area was normalized to the area of the spinal cord segment spanning the injured core in a 4 × image. To evaluate axon regeneration, the immunoreactivity of 5-HT was normalized to the area of the spinal cord segment spanning the injured core in a 10 × image, and the number of NF + axons longer than 1 μm in the GFAP − region was counted and normalized to the area of the GFAP − region. For each sample, sections spanning the injured core and two adjacent sections spaced 180 μm apart were quantified, and the results from each section were averaged, with 5 samples per group.
To evaluate the proliferation of fibroblasts, BrdU + PDGFRβ + or Ki67 + PDGFRβ + cells were counted on 40 × images spanning the injured core. The average of three random 40 × images was used as the final result of each sample, with 5 samples per group.

Behavioural assessments
The Basso Mouse Scale (BMS) is widely used to evaluate locomotor function recovery after SCI in mice [18]. In this study, BMS was performed in an open field according to the protocol developed by Basso and colleagues [19]. All mice received BMS to confirm normal locomotor function before SCI and received BMS to confirm the success of the SCI model after surgery. Each mouse was assessed by two experienced examiners at 3, 7, 14, 21 and 28 dpi, and the average value was finally obtained, with 8 animals per group.
Footprint analysis was used to further evaluate locomotor function recovery at 28 dpi and was performed according to previous reports [20]. The mice without SCI received footprint analysis were included in the uninjured group. The front paws were dipped in green dyes, and the hind paws were dipped in red dyes. The stride length was determined by the distance from the beginning to the end of the hind paw in a step. The stride width was determined by the distance from the outermost toe of the left paw to the outermost toe of the right paw. The paw rotation was determined by the angle between the midline axis of the body and the axis of the hind paw. All assessments were performed in three consecutive gait cycles on each side and averaged, with 8 animals per group.
All behavioural assessments were performed in a blind fashion.

Statistical analysis
The data are presented as the mean ± standard error of the mean (SEM), and individual data points are plotted in the figures. The statistical methods used are presented in the figure legends. Multiple comparisons were analysed with one-way or two-way analysis of variance (ANOVA) with a post hoc Tukey-Kramer test, and comparisons between two groups were performed using Student's t test. Data analysis and chart production were performed using GraphPad Prism 8.0 (GraphPad, United States), and a value of p < 0.05 was considered statistically significant.

Different spatiotemporal distributions of PDGFB and PDGFD after SCI
After SCI, PDGFRβ is expressed in all fibrotic scarforming fibroblasts [13], and PDGFRβ can only be activated by PDGFB and PDGFD to participate in a variety of biological processes, including peripheral organ fibrosis [10][11][12]. We speculated that PDGFRβ is involved in the formation of fibrotic scar after SCI. Therefore, immunofluorescent staining was performed to confirm the spatiotemporal distribution of PDGFB, PDGFD and PDGFRβ after SCI. The results showed that the PDGFRβ + fibroblasts increased significantly and aggregated gradually to the injured site at 3 to 7 dpi, while a contiguous fibrotic scar boundary formed to corral the injured core at 14 to 28 dpi (Figs. 1, 2), which was consistent with previous studies [4][5][6]. Meanwhile, PDGFB and PDGFD were widely expressed and distributed adjacent to PDGFRβ after SCI until 28 dpi (Figs. 1, 2). These results suggest that there may be an interaction between the ligand PDGFB or PDGFD and the receptor PDGFRβ, which may be involved in fibrotic scar formation after SCI. Interestingly, PDGFB was significantly expressed around the lesion epicentre from 7 dpi (Fig. 1I-T), while PDGFD was expressed earlier from 3 dpi (Fig. 2E-H) and preferentially distributed at the lesion epicentre at 14-28 dpi (Fig. 2M-T). These results suggest that PDGFB and PDGFD may have different cell sources after SCI.

PDGFB is mainly secreted by astrocytes, while PDGFD is mainly secreted by macrophages/microglia and fibroblasts after SCI
To preliminarily explore the cell sources of PDGFB and PDGFD, we detected their costaining with the main cell components of the injury site, including macrophages/ microglia, fibroblasts, astrocytes and vascular endothelial cells. GFAP was used to label astrocytes, CD31 was used to label vascular endothelial cells, and CD68 was used to label macrophages/microglia. The staining results showed substantial colocalization between PDGFB and GFAP + astrocytes or PDGFRβ + fibroblasts at 14 dpi ( Fig. 3A-H). GFAP + PDGFB + cells and PDGFRβ + PDGFB + cells accounted for 83.26 ± 1.56% and 13.69 ± 0.85% of PDGFB + cells, respectively (Fig. 3Q). There was no significant colocalization between PDGFB and CD68 + macrophages/microglia or CD31 + vascular endothelial cells ( Fig. 3I-P). Furthermore, GFAP + PDGFB + cells and PDGFRβ + PDGFB + cells were adjacent to each other at the edge of the injured core (Fig. 3H). These results indicate that PDGFB is mainly secreted by astrocytes after SCI.

In situ injection of PDGFB or PDGFD induces fibrosis in the uninjured spinal cord
To directly investigate the effect of the PDGFRβ pathway, a single factor, on fibrotic scar formation after SCI, we injected exogenous PDGFB or PDGFD into the uninjured spinal cord to activate the PDGFRβ pathway. Immunofluorescence staining was used to detect PDGFRβ, fibronectin and laminin to observe the changes in fibroblasts and fibrous ECM. The results of the control group showed that the injection itself did not lead to PDGFB or PDGFD expression and fibroblast aggregation (  ( Fig. 5D-F, J-L), while PDGFD was mainly located at the fibrosis core in the uninjured spinal cord (Fig. 5P-R, V-X). Therefore, our results indicate that the activation of the PDGFRβ pathway by PDGFB or PDGFD can directly induce fibrosis in the uninjured spinal cord.
To further verify the specific role of the PDGFRβ pathway in fibrotic scar formation, intrathecal injection of SU16f was used to block the activation of PDGFRβ in the uninjured spinal cord that received the injection of PDGFB or PDGFD. SU16f is a potent and highly selective PDGFRβ inhibitor that displays > 14-fold, > 229-fold and > 10,000-fold selectivity over VEGFR2, FGFR1 and EGFR, respectively, and SU16f has been used to specifically block PDGFRβ [21][22][23][24]. In our study, SU16f was preinjected the day before the injection of PDGFB or PDGFD and then injected daily for 7 consecutive days from the day of PDGFB or PDGFD injection. Our results showed that the fibrosis, as determined by PDGFRβ + , fibronectin + and laminin + staining, was significantly reduced in the uninjured spinal cord of the mice that received the combined injection of SU16f and PDGFB or PDGFD (Fig. 5B'-D' , H'-J') compared with that of the mice that received the injection of PDGFB or PDGFD alone (Fig. 5Y-A' , E'-G'). These results indicate that the PDGFRβ pathway inhibitor SU16f can block exogenous PDGFB-or PDGFD-induced fibrosis in the uninjured spinal cord. Interestingly, SU16f completely blocked PDGFD-induced fibrosis, but only partially blocked PDGFB-induced fibrosis. These results suggest that PDGFB and PDGFD may be involved in different phases of fibrotic scar formation, and we emphasize that the process and mechanism are worthy of in-depth study. Overall, excluding the influence of the complex microenvironment after SCI, we used this method to more specifically confirm that the PDGFRβ pathway is a sufficient factor for fibrosis.

Intrathecal injection of the PDGFRβ blocker SU16f inhibits fibroblasts proliferation and fibrotic scar formation after SCI
To further confirm the role of the PDGFRβ pathway in regulating fibrotic scar formation after SCI, intrathecal injection of SU16f was used to treat the mice with SCI. The injured spinal cord is in the period of apoptosis and necrosis at 3 dpi, from which fibroblasts begin to proliferate and aggregate in the injured site [5]. Therefore, we performed daily intrathecal injection of SU16f from 3 dpi to block the PDGFRβ pathway (Fig. 10A). Immunofluorescence staining showed that the fibrotic scar, manifested as the PDGFRβ + , fibronectin + and laminin + areas, was significantly reduced at 28 dpi after the intrathecal injection of SU16f compared with the control group (Fig. 6). These results indicate that SU16f blockade of the PDGFRβ pathway can inhibit the formation of fibrotic scar after SCI, resulting in a reduction in fibrotic scar area.
It has been reported that the number of fibroblasts reaches its peak at 7 dpi, which is mainly caused by the proliferation of fibroblasts inherent in the spinal cord, suggesting that fibroblasts proliferation is an important process of fibrotic scar formation after SCI [4]. Therefore, immunofluorescence staining detecting BrdU and Ki67 was used to evaluate the effect of SU16f on the proliferation of fibroblasts at 7 dpi. The results showed that the density of BrdU + PDGFRβ + cells (Fig. 7A-H, Q) and Ki67 + PDGFRβ + cells ( Fig. 7I-P, R) in the SU16f group was significantly lower than that of the control group at 7 dpi. These results suggest that SU16f blockade of the PDGFRβ pathway can inhibit the proliferation of fibroblasts after SCI, which may contribute to the reduction in fibrotic scar.

Intrathecal injection of SU16f breaks the fibrotic/ astrocytic scar boundary, shrinks the lesion and inhibits inflammation after SCI
Following SCI, astrocytic scar and fibrotic scar form a dense and contiguous boundary surrounding the injured core, which is one of the important reasons for the failure of axon regeneration [1,6,17]. Therefore, we further explored the effect of SU16f blockade of PDGFRβ on the fibrotic/astrocytic scar boundary, lesion size and inflammation after SCI. GFAP was used to label astrocytic scar and CD68 was used to label inflammatory cells after SCI. The results showed that compared with the control group, the GFAP − area indicated that the lesion size was significantly reduced at 28 dpi after the intrathecal injection of SU16f (Fig. 8A-H,  Q). The contiguous boundary of the fibrotic/astrocytic scar was oriented parallel to the injured core at 28 dpi in the control group (Fig. 8D, L). However, after intrathecal injection of SU16f, the astrocytic/fibrotic scar was disordered and was oriented perpendicular to the injured core at 28 dpi (Fig. 8H, P). Therefore, our results indicate that SU16f blockade of the PDGFRβ pathway breaks the fibrotic/astrocytic scar boundary and shrinks the lesion after SCI. Breaking of the scar barrier may facilitate the passage of regenerated axons through the injured core, which has been assessed below.
It has been reported that fibrotic scar corrals inflammatory cells to the injured core after SCI, contributing to limiting inflammation [6,17]. However, compared with the control group, SU16f inhibition of fibrotic scar formation led to a significant decrease in the CD68 +  inflammatory cell area at 28 dpi ( Fig. 8I-P, R). These results suggest that moderate inhibition of fibrotic scar by SU16f blockade of the PDGFRβ pathway contributes to the reduction in inflammation in the chronic phase of SCI.

Intrathecal injection of SU16f promotes axon regeneration and locomotor function recovery after SCI
To further confirm whether the PDGFRβ pathway can be used as a therapeutic target for SCI and the effect of SU16f on axon regeneration after SCI, immunofluorescence staining was used to assess the regeneration of NF + or 5-HT + axons. The GFAP − area was used to distinguish the injured core. The results showed that compared with the control group, the NF + axon density of the injured core in the SU16f group increased significantly after SCI ( Fig. 9A-H, R). In addition, SU16f significantly increased the area of 5-HT + axons of the injured site after SCI ( Fig. 9I-P, S). Three out of 5 mice in the SU16f group presented regenerated 5-HT + axons that passed through the injured core to the caudal side after SCI, which was not observed in the control group (Fig. 9J, M and Q). These results indicate that SU16f blockade of the PDGFRβ pathway contributes to axon regeneration after SCI, which may be caused by reduced fibrotic scar and inflammation (Fig. 8). Furthermore, BMS score and footprint analysis were used to analyse the recovery of locomotor function after SCI. Compared with the mice in the control group, the mice injected with SU16f obtained better hind limb locomotor function at 14, 21 and 28 dpi, corresponding to a higher BMS score (Fig. 10B). In addition, footprint analysis further revealed that the mice in the SU16f group obtained better locomotor function at 28 dpi (Fig. 10C), including longer stride length, shorter stride width and smaller paw rotation, than the mice in the control group ( Fig. 10D-F). Although the mice in the SU16f group obtained better treatment effects, their locomotor function did not return to the level of the uninjured group (Fig. 10C-F). Overall, our results indicate that blocking PDGFRβ with SU16f contributes to the recovery of locomotor function after SCI.

Discussion
In this study, we found that the expression of PDGFD occurred earlier than that of PDGFB after SCI, and PDGFB was mainly secreted by astrocytes, while PDGFD was mainly secreted by macrophages/microglia and fibroblasts. Moreover, in situ injection of exogenous PDGFB or PDGFD can lead to fibrosis in the uninjured spinal cord, while SU16f blockade of the PDGFRβ pathway reduced the fibrotic scar area, interrupted the fibrotic/astrocytic scar boundary, shrunk the lesion and inhibited inflammation, promoting axon regeneration and locomotor function recovery after SCI. Therefore, the PDGFRβ pathway is expected to be a therapeutic target after SCI.
SCI is a devastating trauma and causes sensory and locomotor dysfunction in patients, and there is currently a lack of effective clinical treatments [25,26]. Therefore, it is of great significance to explore the pathological changes and molecular mechanisms of SCI, so as to provide new ideas for treatment. Scars, as one of the critical factors hindering axon regeneration after SCI, mainly include fibrotic scar formed by fibroblasts and astrocytic scar formed by astrocytes [1,5]. Although the deposition of chondroitin sulphate proteoglycans (CSPGs) by astrocytes leads to the failure of axon regeneration after SCI [27,28], the secretion of axon-growth-supporting molecules by astrocytes is required for axon regeneration and astrocytic scar has the beneficial effect of limiting inflammation [29,30]. Meanwhile, recent studies have shown that inhibition of astrocytic scar formation cannot promote axon regeneration, while astrocytic scar formation aids rather than prevents axon regeneration [29,31], suggesting that the beneficial effects of astrocytic scar in SCI are greater than the adverse effects. However, moderate inhibition of fibrotic scar formation can promote axon regeneration and functional recovery after SCI [6,17], indicating that fibrotic scar is of great significance as a therapeutic target for SCI.
Following SCI, perivascular fibroblasts leave blood vessels, proliferate and migrate to the injured site at 3-7 dpi [4,5]. At 7-14 dpi, fibroblasts deposit large amounts of fibrous ECM, including fibronectin, laminin and collagen, to form fibrotic scar that corrals macrophages in the injured core and is located on the inner side of astrocytic scar [7,17,18]. It has been accepted that fibrotic scar significantly hinders axon regeneration after SCI [8,[32][33][34]. The Jonas Frisén group used Glast-Rasless transgenic mice to specifically block the proliferation of fibroblasts after SCI, thereby establishing a fibrotic scar removal model [4,6]. In addition, they revealed that complete elimination of fibrotic scar leads to the failure of injured site closure and the spread of inflammation after SCI, while a moderate reduction in fibrotic scar inhibits inflammation and promotes axon regeneration after SCI [4,6], suggesting that fibrotic scar can be used as a therapeutic target after SCI. However, previous reports mainly used transgenic strategies or nonspecific target intervention strategies to inhibit fibrotic scar formation after SCI [6,8,32]. For instance, transforming growth factor beta (TGF-β) not only is a profibrotic factor but also participates in a variety of biological processes [35,36]. Administration of 8-Br-cAMP, Taxol, epothilone B (epoB) or antagomir-21 has been successfully used to suppress fibrotic scar after SCI via inhibiting TGFβ pathway, while targeting TGFβ is not specific for regulating fibrotic scar [32][33][34]37]. Therefore, a better understanding of the molecular mechanism of fibrotic scar formation after SCI could lead to the uncovering of specific molecular therapeutic targets, which is of major significance.
PDGFRβ is a transmembrane receptor tyrosine kinase composed of an intracellular tyrosine kinase domain and an extracellular ligand binding domain [9]. PDGFB or PDGFD binding to PDGFRβ monomers mediates dimerization of PDGFRβ monomers and then activates kinase activity to trigger intracellular signalling cascades, including Janus kinase (JAK), phospholipase C gamma (PLCγ) and phosphoinositide 3-kinase (PI3K), involved in cell proliferation, differentiation and migration [10,11]. It has been reported that PDGFRβ is expressed in pericytes, astrocytes, NG2 cells and endothelial cells in brain injury models [38]. However, PDGFRβ is widely used to label pericytes and regulates the survival, proliferation and migration of pericytes in the brain, thereby participating in angiogenesis and the repair and maintenance of the blood-brain barrier (BBB) [12]. In the injured spinal cord, PDGFRβ is expressed on all fibrotic scar-forming fibroblasts, and fibroblasts account for up to 95% of PDGFRβ + cells, indicating that PDGFRβ is specifically expressed in fibroblasts after SCI [7,8,13]. However, the spatiotemporal distribution of the ligands PDGFB and PDGFD and the effect of activation of the PDGFRβ pathway on fibroblasts forming fibrotic scar after SCI remain unclear. In this study, we found that PDGFB and PDGFD were highly expressed and distributed adjacent to PDGFRβ after SCI, suggesting that PDGFB or PDGFD may activate PDGFRβ to be involved in the formation of fibrotic scar after SCI. In addition, PDGFB began to be expressed in a large area from 7 dpi and was gradually distributed around the lesion epicentre, while PDGFD Fig. 10 Intrathecal injection of SU16f promotes locomotor function recovery after SCI. A Timeline of the intrathecal injection and the behavioural assessment. B Locomotor function was evaluated by BMS at 0, 3, 7, 14, 21 and 28 dpi. ***P < 0.001 and ****P < 0.0001 vs. Control group by two-way ANOVA, n = 8 animals per group. C Representative images of footprint analysis in the Uninjured, Control and SU16f groups at 28 dpi. The front paws are shown in green dyes, and the hind paws are shown in red dyes. D-F Quantification of the stride length, stride width and paw rotation at 28 dpi. *P < 0.05, ***P < 0.001 and ****P < 0.0001 by one-way ANOVA, n = 8 animals per group began to be expressed in a large area from 3 dpi and was gradually distributed at the lesion epicentre. These results indicate that PDGFD may be mainly involved in the recruitment and proliferation of fibroblasts in the early stage, while PDGFB may be mainly involved in the assembly and maturation of fibrotic scar in the late stage. The functional difference between PDGFB and PDGFD needs to be further studied, which is expected to provide a theoretical basis for sequential intervention of the PDGFRβ pathway after SCI.
Fibroblasts, astrocytes, vascular endothelial cells and macrophages/microglia are important cellular components at the injured site of SCI, and recent evidence has demonstrated extensive crosstalk among them [17,18,39,40]. The inhibition of fibrotic scarring results in the attenuation of astrogliosis and the interruption of astrocytic scar boundary after SCI [6]. Besides, the inhibition of astrocytic scarring leads to the interruption of fibrotic scar boundary and the spread of inflammation after SCI [39,41], and the depletion of macrophages or microglia in the injured core leads to the interruption of fibrotic scar boundary after SCI [17,18,42]. However, the molecular mechanism cues for the crosstalk among the cells remain largely elusive. Therefore, we further investigated the cell sources of PDGFB and PDGFD and focused the sources on macrophages, astrocytes, vascular endothelial cells or fibroblasts, which was expected to provide a basis for the crosstalk among the main cell components at the injured site. Our results showed that PDGFB was mainly secreted by astrocytes, while PDGFD was mainly secreted by macrophages/microglia and fibroblasts after SCI. The different sources of PDGFB and PDGFD indicate their different functions after SCI, while whether the PDGF/PDGFRβ pathway plays a role in the crosstalk among astrocytes, macrophages/microglia and fibroblasts needs to be further investigated.
To directly explore the effect of the PDGFRβ pathway, a single factor, on fibrotic scar formation, we injected exogenous PDGFB or PDGFD into the uninjured spinal cord instead of the injured spinal cord to avoid the influence of the complex microenvironment of SCI. Our results showed that both PDGFB and PDGFD can promote fibrosis in the uninjured spinal cord, and the profibrotic effect can be blocked by the PDGFRβ inhibitor SU16f. The results of FN-or LN-labelled fibrosis was consistent with those of PDGFRβ-labelled fibrosis. Therefore, our results were reliable and preliminarily confirmed that the activation of the PDGFRβ pathway is sufficient to induce fibrosis. Notably, SU16f completely blocked PDGFDinduced fibrosis but only partially blocked PDGFBinduced fibrosis in the uninjured spinal cord, suggesting that PDGFB and PDGFD may be involved in different phases of fibrotic scar formation. We emphasize that the process and mechanism are worthy of in-depth study. In addition, SU16f blockade of the PDGFRβ pathway was performed to further confirm the effect of the PDGFRβ pathway on fibrotic scar formation after SCI. The results showed that SU16f significantly inhibited the proliferation of fibroblasts and reduced fibrotic scar after SCI. Therefore, our results provide direct evidence that the PDGFRβ pathway mediates fibrotic scar formation after SCI, which can be blocked by SU16f inhibiting the proliferation of fibroblasts.
The dense contiguous fibrotic/astrocytic scar boundary is an important component of the inhibitory microenvironment after SCI [1]. The physical barrier of the scars directly prevents the regenerated axons from passing through the injured core, and the axon tips form retraction bulbs after contacting fibrotic scar, resulting in the failure of axon regeneration after SCI [6]. Therefore, the interruption of the contiguous scar boundary contributes to axon regeneration [6]. In our study, the results showed that SU16f blockade of the PDGFRβ pathway resulted in the interruption of the fibrotic/astrocytic scar boundary and the reduction of the lesion size after SCI, facilitating the regeneration of NF + or 5-HT + axons that passed through the injured core. Interestingly, our results showed that SU16f-induced reduction in fibrotic scar led to a smaller area of inflammatory cells at 28 dpi. The Jonas Frisén group used Glast-Rasless transgenic mice to completely eliminate fibrotic scar after SCI, leading to the spread of inflammatory cells at 14 dpi. However, a moderate reduction in fibrotic scar did not lead to the spread of inflammatory cells at 14 dpi but led to a reduction in inflammatory cells at 28 dpi [4,6]. Therefore, our results are consistent with the results of the Jonas Frisén group, together indicating that moderate inhibition of fibrotic scar after SCI does not lead to the spread of inflammation in the early stage but inhibits the spread of inflammation in the late stage, which contributes to axon regeneration. Blood-derived macrophages migrate towards high concentrations of complement component C5a in the injured core after SCI, and C5a may be secreted by PDGFRβ + fibroblasts [43], suggesting that C5a may be involved in fibroblasts corralling macrophages in the injured core. The effect of fibrotic scar changes on inflammatory response after SCI and its molecular mechanism need to be further investigated. Overall, our results further reveal that the adverse effects of excessively deposited fibrotic scar are greater than its beneficial effects in SCI and can be used as a therapeutic target after SCI. Finally, the results of BMS score and footprint analysis confirmed that SU16f blockade of the PDGFRβ pathway promotes locomotor function recovery in injured mice. Although fibrotic scar forms after SCI in both rats and mice [7], SCI in rats leads to cavity formation in the injured core, which is considered to resemble the pathological changes in patients with SCI in the clinic [44,45]. Our findings should be further validated in rat models, which could contribute to a better understanding of fibrotic scar as a therapeutic target for SCI in clinic. Thus far, specific therapeutic targets for inhibiting fibrotic scar formation after SCI have rarely been reported, and the present study is expected to provide a novel idea.

Conclusion
The present study reveals that PDGFD and PDGFB increase successively after SCI and can activate PDGFRβ + fibroblasts. PDGFD is mainly secreted by macrophages/ microglia and fibroblasts and distributed at the lesion epicentre, while PDGFB is mainly secreted by astrocytes and distributed around the lesion epicentre. Intrathecal injection of the PDGFRβ inhibitor SU16f blocked the fibrosis induced by exogenous PDGFB or PDGFD in the uninjured spinal cord. Furthermore, blocking the PDGFRβ pathway with SU16f reduces fibrotic scar, interrupts scar boundary and inhibits lesion and inflammation, promoting axon regeneration and locomotor function recovery after SCI. This study confirms that the PDGF/PDGFRβ pathway plays a critical role in fibrotic scar formation after SCI and is expected to be a specific target for the treatment of SCI.